Project Summary Recent studies from our group demonstrate that human embryonic stem cell-derived cardiomyocytes (hESC- CM) can improve the function of infarcted hearts of macaque monkeys. These improvements of as much as 20 ejection fraction points are associated with robust remuscularization, often giving centimeter-scale grafts that are visible by MRI. As we progress toward clinical trials, however, several outstanding questions remain unanswered. What is the mechanism of hESC-CM action? How can the relatively low efficiency of cardiac engraftment be improved? Will we get more complete regeneration if we add key myocardial cell types in addition to cardiomyocytes? In Aim 1 we address whether the mechanism of hESC-CM action is related to direct cell replacement, or if there is a significant paracrine component. To test this, we have used CRISPR- Cas9 to delete cardiac and skeletal TNNI genes in hiPSCs, yielding non-contractile cardiomyocytes with intact myofibrils, action potentials and calcium transients. These ?paracrine-only? cardiomyocytes will be compared to wild type cells for their ability to repair the infarcted rat heart. In Aim 2 we will test the hypothesis that a ?smart hydrogel?, designed to signal through the Notch pathway, can improve cardiac regeneration with hiPSC- CMs. This Notch gel stimulates hiPSC-CM proliferation after engraftment and promotes vascular ingrowth from the surrounding host microcirculation. We will test if these structural benefits are accompanied by enhanced ventricular function. Finally, Aim 3 follows up on recently completed studies in the rat, where we observed that hESC-derived epicardial cells (hESC-Epi) are synergistic with hESC-CMs in terms of promoting enhanced remuscularization and functional recovery of the infarcted heart. We will utilize our macaque monkey model to test whether hESC-Epi augment hESC-CM-based heart regeneration, with the hypothesis that these cells will promote hESC-CM maturation and enhance their proliferation, resulting in less arrhythmogenic grafts that more completely remuscularize the infarct. Studies in this proposal will impact directly on our upcoming clinical trials of cardiac repair.